IAEA-TECDOC-252 PROGRAMS PROCESSING RADIOIMMUNOASSAY PROGRAMMABLE CALCULATOR Off-Line Analysi f Countinso g Data from Standard Unknownd san s A TECHNICAL DOCUMENT ISSUEE TH Y DB INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1981 PROGRAM DATR SFO A PROCESSIN RADIOIMMUNOASSAN I G Y USIN HP-41E GTH C PROGRAMMABLE CALCULATOR IAEA, VIENNA, 1981 PrinteIAEe Austrin th i A y b d a September 1981 PLEASE BE AWARE THAT ALL OF THE MISSING PAGES IN THIS DOCUMENT WERE ORIGINALLY BLANK The IAEA does not maintain stocks of reports in this series. However, microfiche copies of these reports can be obtained from INIS Microfiche Clearinghouse International Atomic Energy Agency Wagramerstrasse 5 P.O.Bo0 x10 A-1400 Vienna, Austria on prepayment of Austrian Schillings 25.50 or against one IAEA microfiche service coupon to the value of US $2.00. PREFACE The Medical Applications Section of the International Atomic Energy Agenc s developeha y d severae th ln o programe us r fo s Hewlett-Packard HP-41C programmable calculator to facilitate better quality control in radioimmunoassay through improved data processing. The programs described in this document are designed for off-line analysis of counting data from standard and "unknown" specimens, i.e., for analysis of counting data previously recorded by a counter. Two companion documents will follow offering (1) analogous programe on-linus r conjunction fo i se n wit suitabla h y designed counter, and (2) programs for analysis of specimens introduced int successioa o f assano y batches from "quality-control pools" of the substance being measured. Suggestions for improvements of these programs and their documentation should be brought to the attention of: Robert A. Dudley Divisio Liff no e Sciences IAEA A-1400 Vienna Austria CONTENTS Chapter I. Overview I - 1 1.1 The programs: objectives and strategy I - 1 5 1- calculatoe Th 1.2 r 7 3 1- 1. Organizatio thesf o e e us note d nan s Chapter II. Basic Ideas in Data Analysis II - 1 11.3 1- I I Non-statistical concepts 9 - I I 11.2 Statistical concepts 1 5 - 11. I 3I Summar philosophf o y datf o y a analysis in these programs 1 - I ChapteII r III. HP-41C e CalculatoUs s it d ran 3 - I 111.II 1 Car calculatof eo r system 111.3 2 - AssemblI II calculatof o y r system 111.3 Operation of calculator without III - 3 stored programs 111.4 Operation of calculator with stored programs III - 21 Chapter IV. Analysis of In-Vitro Assay Counting Data IV - 1 o Irregularitie(N Datan si ) IV.1 Programs and functions IV - 1 5 - IV.V I 2 Preparatio calculatof no r IV.3 5 Natur - organizatiod V I ean samplef no s IV.4 Example of data analysis (no IV - 11 irregularitie datan i s ) Chapter V. Analysis of In-Vitro Assay Counting Data V - 1 (Irregularitie Datan si ) V.I Initialization of conditions for standards V - 1 (Functio) nSI V.2 Function SA V - 1 V.3 Function SC V - 9 1 2 - V 4 V. Functio I nU V.5 Function UA V - 23 V.6 Appraisal of quality of results V - 27 7 V. Alternativ7 2 - V e adjustmen shapf o t f eo standard curve Chapter VI. Supplementary Functions for Analysis of VI - 1 In-Vitro Assay Counting Data VI.1 Determination of constants A and B (Program AB) VI - 1 9 - I V VI.2 Functio P nC Appendi Glossar- 1 x Symbolf o y Calculaton i s 1 - rl A Display and Printout Appendix 2 - Chi-Square ( ^C2) Test and Tables A2 - 1 Appendix 3 - Variance-Ratio Test and Tables A3 - 1 1 - 4 A Appendi Bar-Cod- x4 Programr fo e s I - l Chapte . I rOvervie w The programs described in these notes provide for analysis, using the Hewlett Packard HP-41C calculator, of counting data collected in radioitnmunoassays or other related in-vitro assays. The immediate reason for their development was to assist laboratories having limited financial resource d seriouan s s problems f qualito y control e programTh . e structurear s d botr "off-linefo h " use, with manual entr f countino y g data inte calculatoth o r through the keyboard, and, in a slightly altered version, for "on-line" use, with automatic data entry from an automatic well scintillation counter originally designed at the IAEA. Only the off-line variant programe th f o describes i s n thesi d e notes e on-linTh . e version will be covered in a companion document. 1. The programs: objectives and strategy The programs determine from appropriate counting data the concentratio f analytno unknown i e n specimens d provid,an e supplementary information about the reliability of these results and the consistency of current and past assay performance. Radioimmunoassay and related procedures are subject to many errors. Som f theseo e arise froe laboratorth m y manipulations themselves; others are basically attributable to the heterogeneity e substanceoth f s being measuree reagentth d an d s used therefor. Special attention must be given to these errors. A part of this scrutiny can be provided through appropriate data analysis, which has only recently become universally accessible as a result of the decreasing cost and increasing power of calculators and computers. * recena n I t pape Finne, r y states: "The programming needs of bioassay and RIA should focus attention on a common misunderstanding. Too often tfhose Finney, D.J., International Statistical Review (1979)1 7 4 . "Bioassa e practicth d an f statisticayeo l inference." 1-3 t statisticianno e ar o wh s state that method f analysio s s and computer programs for their use should be short and very simple, leaving sophisticated techniques for professional statisticians n realityI . r analysefo , s wanted frequently in routine processing of data, the opposite is nearer to the truth. The experienced statistician who scans his data carefully often senses the occurrence of non-linearities, outliers, variance heterogeneities and the like without requiring dependence n speciao l computation d testssan A clinica. l biochemist is less well equipped for this type of data appreciation, and, very properly n othe o mins ,s hi i rd features hi f o s problem neede ;h protectioe th s a sophisticate f o n d program that employs many component r monitorinfo s e th g estimatione th r datneede h fo wels a d s sa l,an full well-formatted output." Finney goes on to regret the common use of computers (or anyway, computer programs) of inadequate power. These program n attempa e meeo ar schallenge t t th t e described by Finney through use of what may be the lowest level of computational power that can be considered defensible today: "lowest" because in view of the other costs of in-vitro assays, acquisitio a les f so n powerful calculato s probabli r n a y insignificant economy; "defensible" becaus e statisticath e l perceptions offere y thesb d e programs substantially exceed those yielde y datb d a processing procedures than e currentli ar t e us n i y most small laboratories. The strategy of data analysis adopted in these programs stems frowore manf th mko y investigators. However reflectt i , a specias l debt to the work of R.P. Ekins, P.G. Malan and colleagues; D. Rodbard, P. Munson and colleagues; and R.P.C. Rodgers. Many of the ideas generate n receni d t years, especially e foregoingth y b , have * been systematized in a recent book by Rodgers that is admirably suited as a supplementary reference for these notes. The central ideas in the strategy here employed are that a paramount role of automatic data processing lie n erroi s r accounting d thae ,an th t Rodgers, R.P.C., Data analysis and quality control in binder-ligand assay, Scientific Newsletters, Anaheim, CA, 1981. 1-5 concep f "imprecisioo t n profile usefua s i "l toor thifo ls purpose. The curve-fitting model adopted (an issue that may not deserve the obsessive attention devoted to it in most discussions of automatic data processing) is the 4-parameter logistic. The fitting is by weighted least-squares procedures, and provision is made for easy adjustmen curvf o t e shap accommodato t e e unusual assays. Since these program y speciala s l stres assessinn so e th g reliabilit e assay assumes th i f t o y,i d tha t leasa te assayison t t in the laboratory is seriously dedicated to detecting, understanding eliminatind ,an g errors e programTh . s will relieve him of much of the labour of data analysis as required for this assessment cannot ,bu t servsubstituta s a e dedicationr fo e e th f I . information yielded by these programs is conscientiously examined, it will yiel benefitso tw d . First wilt i , l draw attentioo t n certain suspect results r examplfo , e unknowns thae unreliablear t , or even whole assay batches that should be discarded. Second, it can provid objectivn a e e mean distinguishinr sfo g betweee nth comparative performance of alternative analytical procedures, i.e., it can help the assayist to improve his procedures. 2. The calculator The HP-41C is a programmable "pocket calculator" with alphanumeric capabilitie attachee b whico t sn hca d various accessories present :a printer-plottera t , magnetic card reader, bar-code reader d assorte,an d memory modules n advance(A . d version recently releasede fulth l s memorHP-4e ha ,th , 1yCV capacite th n i y calculator itself.